scholarly journals Structural basis for ion selectivity in TMEM175 K+ channels

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Janine D Brunner ◽  
Roman P Jakob ◽  
Tobias Schulze ◽  
Yvonne Neldner ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ph Regulation ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channels ◽  
X Ray ◽  
Lysosomal Ph ◽  
Additional Layer ◽  
Channel Opening

The TMEM175 family constitutes recently discovered K+channels that are important for autophagosome turnover and lysosomal pH regulation and are associated with the early onset of Parkinson Disease. TMEM175 channels lack a P-loop selectivity filter, a hallmark of all known K+ channels, raising the question how selectivity is achieved. Here, we report the X-ray structure of a closed bacterial TMEM175 channel in complex with a nanobody fusion-protein disclosing bound K+ ions. Our analysis revealed that a highly conserved layer of threonine residues in the pore conveys a basal K+ selectivity. An additional layer comprising two serines in human TMEM175 increases selectivity further and renders this channel sensitive to 4-aminopyridine and Zn2+. Our findings suggest that large hydrophobic side chains occlude the pore, forming a physical gate, and that channel opening by iris-like motions simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen.

scholarly journals Structural basis for ion selectivity in TMEM175 K+ channels

2018 ◽  
Author(s):  
Janine D. Brunner ◽  
Roman P. Jakob ◽  
Tobias Schulze ◽  
Yvonne Neldner ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Structural Basis ◽  
Channel Pore ◽  
K Channels ◽  
X Ray ◽  
Selective Filter ◽  
Channel Opening ◽  
Conformational Rearrangements ◽  
Pore Lining ◽  
Conductive State

AbstractThe TMEM175 family constitutes recently discovered K+ channels that lack signatures for a P-loop selectivity filter, a hallmark of all known K+ channels. This raises the question how selectivity in TMEM175 channels is achieved. Here we report the X-ray structure of a bacterial TMEM175 family member in complex with a novel chaperone built of a nanobody fusion-protein. The structure of the channel in a non-conductive conformation was solved at 2.4 Å and revealed bound K+ ions along the channel pore. A hydrated K+ ion at the extracellular pore entrance that could be substituted with Cs+ and Rb+ is coordinated by backbone-oxygens forming a cation-selective filter at the tip of the pore-lining helices. Another K+ ion within the pore indicates the passage of dehydrated ions. Unexpectedly, a highly conserved threonine residue deeper in the pore conveys the K+ selectivity. The position of this threonine in the non-conductive state suggests major conformational rearrangements of the pore-lining helices for channel opening, possibly involving iris-like motions.

scholarly journals Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 729-739 ◽  
Author(s):  
Yu-Yang Zhang ◽  
Janice L. Robertson ◽  
Daniel A. Gray ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Single Amino Acid ◽  
Structural Basis ◽  
K Channels ◽  
Chord Conductance ◽  
In Series ◽  
Carboxy Terminal

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at Vm = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba2+, suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl+:K+, accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K+ transport.

scholarly journals Mutations reveal voltage gating of CNGA1 channels in saturating cGMP

2009 ◽  
Vol 134 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Open Probability ◽  
K Channels ◽  
Voltage Gated ◽  
Open Conformation ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Low Probability ◽  
Inherent Voltage

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K+ channels, not the S4-based mechanism of voltage-gated K+ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.

scholarly journals A pharmacological master key mechanism that unlocks the selectivity filter gate in K+channels

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. 875-880 ◽  
Author(s):  
Marcus Schewe ◽  
Han Sun ◽  
Ümit Mert ◽  
Alexandra Mackenzie ◽  
Ashley C. W. Pike ◽  
...  
Keyword(s):  
Selectivity Filter ◽  
Rational Drug Design ◽  
Related Gene ◽  
K Channels ◽  
X Ray ◽  
Voltage Gated ◽  
X Ray Crystallography ◽  
Rational Drug ◽  
Dynamics Simulations ◽  
Pore Domain

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+channels gated at their selectivity filter (SF), including many two-pore domain K+(K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+channel activators and highlight a filter gating machinery that is conserved across different families of K+channels with implications for rational drug design.

scholarly journals Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

2015 ◽  
Vol 112 (50) ◽  
pp. 15366-15371 ◽  
Author(s):  
Joshua B. Brettmann ◽  
Darya Urusova ◽  
Marco Tonelli ◽  
Jonathan R. Silva ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Coupling ◽  
Dynamic Coupling ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Dependent Inactivation ◽  
Ion Binding Sites ◽  
Essential Connection

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

scholarly journals Structural basis of ion permeation gating in Slo2.1 K+ channels

2013 ◽  
Vol 142 (5) ◽  
pp. 523-542 ◽  
Author(s):  
Priyanka Garg ◽  
Alison Gardner ◽  
Vivek Garg ◽  
Michael C. Sanguinetti
Keyword(s):  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Ion Permeation ◽  
Channel Activation ◽  
K Channels ◽  
Voltage Gated ◽  
Channel Function ◽  
Activation Gate

The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.

scholarly journals Author response: Structural basis for ion selectivity in TMEM175 K+ channels

2020 ◽  
Author(s):  
Janine D Brunner ◽  
Roman P Jakob ◽  
Tobias Schulze ◽  
Yvonne Neldner ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Author Response ◽  
Structural Basis ◽  
K Channels

scholarly journals Extracellular protons titrate voltage gating of a ligand-gated ion channel

2010 ◽  
Vol 136 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Low Ph ◽  
Extracellular Ph ◽  
Selectivity Filter ◽  
Voltage Gated ◽  
Cyclic Nucleotide Gated Channels ◽  
Open Conformation ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Electric Signals

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.

scholarly journals Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels

2015 ◽  
Vol 146 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Yee Ling Lam ◽  
Weizhong Zeng ◽  
Mehabaw Getahun Derebe ◽  
Youxing Jiang
Keyword(s):  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
Structural Difference ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Studies ◽  
Cyclic Nucleotide Gated Channels ◽  
Cng Channel ◽  
High Resolution Structure ◽  
Calcium Permeability

Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca2+ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2+ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca2+ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca2+ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.

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